Because of the extraordinary complexity of the serum proteome, tumor biomarker discovery by direct proteomic analysis of blood serum continues to be a challenge. As an alternative, this application proposes to analyze proteins secreted from tumor cells in culture and later validate these candidate tumor markers using serum samples from tumor patients and healthy controls. The central hypothesis is that tumor biomarkers can be identified as proteins displaying enhanced secretion upon loss of a tumor suppressor. The objective of the present application is to test this hypothesis using the von Hippel-Lindau (VHL) tumor suppressor as a model case: it is directed towards identifying the VHL tumor biomarkers as proteins displaying increased secretion upon loss of the VHL tumor suppressor. VHL disease is a hereditary cancer syndrome characterized by clear cell renal carcinomas, hemangioblastomas of the nervous system, and adrenal pheochromocytomas. Patients with VHL disease harbor one wild-type VHL allele and one inactivated VHL allele. Tumors and cysts in these patients arise by somatic mutation of the remaining wild-type allele. Biallelic VHL inactivation is also common in sporadic clear cell renal carcinomas and hemangioblastomas Currently, there is no effective serum tumor biomarker for the VHL disease or renal cell carcinomas. Such biomarker would facilitate the monitoring and pre-clinical intervention of VHL mutation carriers and early detection of sporadic renal carcinomas. Employing a quantitative proteomics technology, ICAT (isotope-coded affinity tag), a preliminary small- scale analysis of protein secretion from a VHL-null renal carcinoma cell line, 786-O, and its VHL-reintroduced derivative was carried out. This analysis identified a number of proteins that display enhanced secretion upon VHL loss, including Vascular Endothelial Growth Factor (VEGF) which showed significantly elevated serum levels in VHL disease patients. Guided by these preliminary data, this application pursues two specific aims: 1) scale-up and extend the preliminary analysis using 3 different cell systems and comprehensively identify candidate VHL tumor biomarkers and 2) validate these candidate markers using serum samples from VHL disease patients and healthy controls and also determine the absolute serum concentration ranges of the validated markers in these patients and controls. PUBLIC HEALTH RELEVANCE: By analyzing proteins secreted upon loss of the VHL tumor suppressor, the proposed project discovers serum biomarkers for VHL-mutated tumors, which will advance the monitoring and pre- clinical intervention of VHL mutation carriers as well as early detection of sporadic renal cell carcinomas. Furthermore, secretome proteomics-based approach for cancer biomarker discovery developed by this project will be widely applicable to other tumor suppressors and oncoproteins, facilitating the identification of many useful cancer biomarkers.